By using a flow cytometric technique which allows direct identification of proliferating cells within mixed cell populations, we have previously described that soluble extracts obtained from Mycobacterium tuberculosis or M. avium represent strong stimuli for humanγδ+ T cells. In the present study, we demonstrate that the protocol used for the preparation of M. tuberculosis soluble extracts may have an impact on their γδ+ T cell stimulatory capacity. In agreement with our previous data, soluble extracts prepared from bacteria killed at 85°C and directly disrupted by prolonged sonication (TBe), elicited a strong proliferation of γδ+ T cells after 6-7 days of stimulation. In contrast, when soluble extracts were obtained from bacteria autoclaved (121°C, 25 min) and then washed by centrifugation, a predominant proportion of CD4+ αβ+ T cells was achieved in the responding population. The stimulatory activity for γδ+ T cells was recovered in the supernatant of the autoclaved bacteria, indicating that autoclaving ofM. tuberculosis bacilli releases an antigen(s) into the supernatant which stimulates human γδ+ T cells. While protease digestion of TBe only partially reduced its stimulatory capacity onγδ+ T cells, the stimulatory component(s) released into the supernatant after autoclavation of bacilli was found to be sensitive to protease digestion. Interestingly, in contrast to the preponderant proportion of γδ+ T cells induced in the responding population by unfractionated TBe, when the extract was fractionated by fast performance liquid chromatography (FPLC), most of the fractions exhibited a strong stimulatory capacity on CD4+ αβ+ T cells only. The γδ+ T cell stimulatory activity was confined to the low molecular weight range FPLC fractions. Such results may suggest a possible regulatory role of γδ+ T cells on CD4+αβ+ T cells.